Stunt Ranch Santa Monica Mountains Reserve

01 Mar, 04 AM - 01 Sep, 05 AM

Toxins play important roles as chemical signals in animal communication, influencing predation 1,2, courtship 3,4,5, fecundity 6, and habitat colonization7. The potent neurotoxin tetrodotoxin (TTX) elicits antipredatory behavior in larvae of the California newt, Taricha torosa, as a response to cannibalistic adults 8 and it has been suggested that TTX operates community wide. The ultimate question of my study is to determine if TTX, as a chemical signal, is able to alter community structure and if so, whether it is of keystone significance. While the keystone species concept relies on a species relative abundance and trophic interactions to predict impacts upon communities, the keystone molecule concept proposes that highly conserved molecules may have similar ecological significance 9. TTX could be of keystone significance since it is introduced into streams by T. torosa in trace amounts as a byproduct of chemical defense; thus it is not ubiquitous. Moreover, keystone species have long been thought of as integral for understanding community composition and structure 10,11, yet few studies show how naturally occurring chemical signals alone affect community dynamics. Based on preliminary studies I conducted in the summer of 2010, the behavior of stream invertebrates appears to be modified in the presence of TTX (Figure 1). These behavioral shifts included avoiding TTX and altering invertebrate foraging patterns (Figures 2 and 3). The effects of TTX from my research suggest a far greater impact within the system at numerous trophic levels than expected. If TTX modifies animal behaviors then the molecule should play an important role in determining stream community structure and composition. More so, the significance of TTX within the community could be greater than the direct ecological interactions (predation or competition) of the organism. For this reason, TTX should be of keystone significance. To test the keystone molecule concept I will characterize field distribution of invertebrates within three watersheds of the Santa Monica Mountains. Access to a 20-year data set of field surveys, knowledge of historical events (e.g. floods, fires, droughts), and established relationships with park service personnel and local students will be invaluable to me during this study. The following experiments will show how chemical defense for T. torosa, as a byproduct in streams, alters community structure of invertebrates. Question 1 - Does TTX serve as a structuring signal for freshwater stream invertebrates. Prediction: TTX, not newts themselves, will alter invertebrate species composition in stream pools. Methods: In the spring of 2010, I conducted a paired field experiment to test for affects of TTX on invertebrates in a perennial stream in the Santa Monica Mountains. Caged newts were paired with caged frogs in stream beds and positioned in the stream overnight (15 hours). Upon returning 20 cm diameters were sectioned off around each cage and invertebrates were identified and tallied. These preliminary data indicate that invertebrates avoid areas with caged newts (Figure 2). In spring of 2012 through 2013, I will use a similar paired design to determine if TTX (not newts) is mediating the response. I will build model newts that will serve as positive and negative controls. Models will disperse crude prey extract, stream water, or TTX. Each experimental model that releases a fluid will flux at an ecologically realistic rate (approximately 4*10-7 mol l-1). Multiple trials will be done simultaneously in various pools within streams, separated by a minimum of 300 meters. Cages for newts and models will be constructed following Ferrer and Zimmer 201111. Stream characteristics will be noted and recorded throughout the season for each year. Innocuous fluorescein dye will be used to characterize flow and artifact of cages used in experiments. Boundary layer physics will be used to describe the distribution of TTX and how invertebrates in experimental areas would experience the chemical signal. Interpretation: If numbers of aquatic invertebrates within the 20 centimeter diameter control areas and experimental areas differ, then TTX could be altering invertebrate community composition. A difference between models dispersing TTX and live newts as compared to models dispersing TTX and filtered water could mean TTX is altering the community level interactions more so than the direct effects of predation and competition by adult newts. Question 2 - Does TTX alter the predatory behavior of predatory stream invertebrates? Prediction: TTX should modify invertebrate movement and refuge use. Methods: Previous experiments that I conducted to observe odonate nymph species response to TTX show that these species move less in the presence of TTX (Figure 1), thus becoming less effective predators (Figure 3). In spring 2011, I will conduct bioassays that test the tolerance of all aquatic invertebrate species within the Santa Monica Mountains to ecologically realistic concentrations of TTX over a duration of 24 hours. Filtered water will be used as a control. Lethality will be assessed by utilizing Doppler flow probes to detect cessation of the invertebrate circulatory system. I will then create dose lethality curves for all aquatic invertebrate species. A catalogue of species affected by the toxin will be prepared. Species that exhibit tolerance to the toxin will be tested further to determine if and how TTX affects behavior. These species will be tested in bioassay chambers that mimic stream conditions, and either TTX, filtered water, or crude prey extract will be introduced as a plume into the chambers. Video equipment and motion analysis software will be used to empirically quantify any behavioral changes. Interpretation: TTX could function as a chemical cue that decreases movement, foraging, and predatory behavior of some stream invertebrates. These experiments could show that exposure to TTX increases survival of prey species indirectly by hindering high order aquatic invertebrate predators like odonates and belostomatids. Thus, the presence of TTX could be a keystone molecule since it would alter multiple trophic interactions. Question 3 - Does TTX impact composition and behavior of stream invertebrates in multiple watersheds? Prediction: 1) Variation of TTX levels of newts in pools within the same stream will differ and this difference will be observed across watersheds. 2) Furthermore, invertebrate communities within pools with newts will differ from pools without newts, not because of predation, but because TTX will modify where invertebrates assemble. Methods: From spring 2011 thru 2013 I will characterize abundance and distribution of invertebrates within pools of three watersheds, noting differences between pools with newts and pools without newts. I will collect data from each stream throughout the season, noting density of newts, flow, pH, dissolved oxygen content, epiphyton/periphyton, total suspended solids, nitrates, temperature, sulfates, and phosphates, and discharge velocity. I will collect skin punches from newts within pools across three watersheds using a 2mm biopsy skin punch tool. Male newts will be anesthetized using methanesulfonic acid salt (tricaine) dissolved at 60 parts per million. GPS coordinates will be collected for each newt. These samples will be collected at the start of the mating season and from mid June to mid July. Using methods adapted from Hannifin 200413, I will use biochemical processes to isolate TTX in skin punches, then use High Performance Liquid Chromatography (HPLC) to determine concentrations of TTX (nanomoles of TTX per milligram of skin) within each skin sample (data sample in Figure 4). These data will be used to determine if a TTX gradient does exist between watersheds and whether there is seasonal variability of TTX levels in newts. I will also take multiple punches from a single newt to determine levels of variation of concentration of TTX within a newt. To determine if TTX and invertebrate composition covary in streams within the Santa Monica Mountains, I will then remove newts from some inhabited pools, fence the pool off, and insert model newts that will disperse TTX, an extract of crude prey, or filtered water for 24 hours. Similarly, models will also be placed in streams without newts. Boundary layer data and flow data will be used to ensure ecologically relevant dispersal of the fluids from models. Manipulated pools will be compared to pools in the same watershed that have not been manipulated. Time series analyses of refuge utilization of invertebrates will be completed. Interpretation: 1) Skin punch data will show variation of TTX levels of newts within pools of the same watershed as well as between watersheds. 2) If field experiments show a significant difference in refuge utilization between invertebrates of newt inhabited (or TTX models) and newt absent pools (filtered water and prey crude extract models) this would suggest that TTX serves as a chemical cue to stream invertebrates. In effect, the toxin mediates the impacts of top level predators, where invertebrates assemble, and thus, balances an array of trophic relationships. As such, the molecule is arguably of keystone significance.

Visit #26598 @Stunt Ranch Santa Monica Mountains Reserve

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Under Project # 24385 | Research

Neurotoxin as a keystone chemical? The community wide consequences of a naturally occurring toxin

reserve_staff - University of California, Davis

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Gary Bucciarelli Mar 1 - Sep 1, 2012 (185 days)

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